Age and bromodeoxyuridine labelling index as prognostic factors in high-grade gliomas treated with surgery and radiotherapy.

AIMS: To determine the prognostic value of proliferative potential and DNA ploidy in 72 brain tumours (36 grade III and 36 grade IV astrocytomas) using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry. MATERIAL AND METHODS: All 72 patients underwent excision, mostly incomplete of the tumour. After surgery, eight patients received conventionally fractionated radiotherapy, 11 patients received accelerated radiotherapy, and 53 patients received hypofractionated radiotherapy. Tumour samples taken during surgery from each patient were incubated in vitro for 1 h at 37 degrees C with BrdUrd using the high pressure oxygen method. The percentage of BrdUrd-labelled cells (BrdUrd labelling index [BrdUrd LI]), and the total DNA content were evaluated: RESULTS: The tumours showed variability in the BrdUrd LI values, which ranged from 0.3 to 19.1%. No difference was observed in mean BrdUrd LI between grade III and grade IV sub-groups. A significantly higher percentage of DNA aneuploidy was observed in grade III gliomas (69.4%) than in grade IV gliomas (52.8%). Univariate analysis showed that younger patients (< or = 51 years) (P = 0.021) with grade III gliomas (P = 0.030) and low tumour proliferation rate (BrdUrd LI < or = 2.7%, P = 0.028) had significantly higher 5-year survival rates. Tumour ploidy had no influence on patients' survival (P = 0.591). However, Cox multi-variate analysis showed that only age over 51 years, and high tumour proliferation rate (BrdUrd LI > 2.7%), were significant unfavourable prognostic factors in patient survival. CONCLUSION: In this study, independent prognostic factors for patients with high-grade gliomas treated with surgery and post-operative radiotherapy are age and tumour proliferation rate assessed according to the BrdUrd LI.

Are stromal fibroblasts from cervical tumors suitable to predict normal tissue radiation reaction?

We examined the effect of gamma-irradiation (4 Gy) alone or combined with estrogen (17beta-estradiol 15 microM) treatment on the radiation response of stromal fibroblasts from cervical tumors. The fibroblasts were derived from tumors of 9 younger (<50 years) and 9 older (>50 years) cervical cancer patients. A normal fibroblast GSH+/+ cell strain was used as a reference cell. The end-points examined 2 days after irradiation were cell cycle distribution and apoptosis as measured of the cellular response to gamma-radiation. The response of examined fibroblast groups to gamma-rays alone was comparable but apoptotic death was more marked in fibroblasts derived from the younger patients with TNM 1+2 tumors than from the older ones. There was a considerable estrogen effect on the response to gamma-rays that differed between stromal fibroblasts from the examined age groups and was dependent on the tumor stage. In particular, we found a marked decrease in the number of apoptotic cells and debris after estrogen + irradiation, as compared to irradiation alone, only in younger patients and TNM 1+2 tumors. These results indicate that the response of stromal fibroblasts to gamma-rays to a considerable extent depends on donors age and tumor stage. Since stromal fibroblasts have been used for prediction of normal tissue late effects in patients treated with radiotherapy, we conclude that they may not be an adequate model for this purpose.

Biologically effective doses in radiotherapy of cervical carcinoma*.

Presented study evaluates biologically effective dose (BED) in patients receiving low-medium dose-rate (LDR/MDR) brachytherapy (BRT) plus external beam radiotherapy (XRT) based on tumor cell proliferation values in cancer of the cervix patients. This study includes 229 patients treated entirely by radiotherapy at the Centre Oncology in Krakow. Doses to Point A were estimated for total treatment for each brachytherapy insertion. BED3 were calculated for reference points in the rectum. The linear quadratic equation was used to calculate BED, which is proportional to log cell kill, and the normalized total dose (NTD), that is, equivalent to a 2 Gy fraction schedule. In BEDs 10 calculation overall treatment time for each patient. Tumor proliferation rate was based on Bromodeoxyuridine labeling index (BrdUrdLI) assessed on biopsy material before beginning the radiotherapy. Total BED at those points was summed for each patient. The medium overall treatment time was 90 days (range 30--210). The mean calculated total BED for point A for tumour and "early reactions" was equal to 104.0 Gy10 and 229.0 Gy3 for the rectum, equivalent to NTD=86.6 Gy and 137.4 Gy in 2 Gy fractions, respectively. Kaplan-Meier analysis revealed that age >50 years, higher than mean BRBEDs and totBEDs doses, gaps in treatments shorter than 40 days and disease free survival (DFS) was significant prognostic factor for overall survival. In the multivariate Cox anaysis age >50 years, BRBED10 >77 Gy and gaps ?40 days appeared to be significant for overall survival. None of the examined parameters was significant for tumor control. However, patients age and shorter gaps in the treatment were predictive for DFS.

A ratio of apoptosis to mitosis, proliferation pattern and prediction of radiotherapy response in cervical carcinoma.

The prognostic significance of apoptotic (AI) and mitotic (MI) indices, and the ratio of these parameters (AI/MI), MIB-1 labeling index (MIB-1LI) and proliferation pattern was studied in 130 (FIGO stage IB-IIIB) squamous cervical cancer patients before radiotherapy. Also the influence of the patients age and tumors pathological features (stage, grade, degree of keratinization) and DNA ploidy on the biological parameters were analysed. AI and MI were assessed on histological sections stained with hematoxylin and eosin, and the MIB-1LI on specimens stained with rabbit anti-human Ki-67 antibody (DAKO Ltd). Sections stained with MIB-1 antibody were used for assessment of the tumor proliferation pattern. The median age of the patients was 55 years (29-80). The median values for MIB-1LI, AI, MI, AI/MI, were: 52.3%, 1.1%, 1.5, and 0.9, respectively. In the univariate analysis median values for cut-off points were used for MIB-1LI, and AI, however, for other parameters significant cut-off points have been chosen. For MI it was 2.6 and for the AI/MI ratio 0.7. The median time of follow-up was 29 months, with a range of 2-145 months. The univariate analysis showed that tumor stage (p=0.7009), grade (p=0.6660) and AI (p=0.9378) had negligible influence on patients survival. However, MI >2.6 (p=0.0442), AI/MI

Comparison of the radiosensitivity of normal-tissue cells with normal-tissue reactions after radiotherapy.

PURPOSE: To investigate whether the in vitro radiosensitivity of normal lymphocytes and fibroblasts evaluated by the micronucleus (MN) assay predicts acute and late reactions after radio-therapy in cancer patients. MATERIALS AND METHODS: Studies were performed on blood samples from 31 cervical and head and neck cancer patients and on skin fibroblasts from eight of the cancer patients. The radiosensitivity of lymphocytes and of fibroblasts was also assessed in 24 and five healthy donors, respectively. Radiosensitivity was measured after in vitro irradiation with doses ranging from 2 to 5 Gy using micronucleus frequency (the number of micronuclei per single binucleated (BN) cell) and the percentage of BN cells with micronuclei. The in vitro results were compared with the maximum grade of acute and late reactions. RESULTS: There was no significant difference in cellular radiosensitivity between cancer patients and healthy donors. Although cancer patients differed considerably in normal-cell radiosensitivity, no correlation was found between radiosensitivity, either of lymphocytes or fibroblasts, and acute and late clinically observed side effects. In addition, no relationship was observed between the radiosensitivity of lymphocytes and fibroblasts derived from the same donors. CONCLUSION: The data do not support the usefulness of the MN assay in predicting normal-tissue response to radiotherapy in cancer patients.

Accumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in porcine preovulatory follicles after in vitro exposure to TCDD: effects on steroid secretion and cell proliferation.

The present experiments were conducted to test 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) accumulation in the tissues, and its influence on cell proliferation and steroid secretion. A dose of 3.2 ng of TCDD/g tissue was added at the beginning of the culture, and the media were changed every 24 h or not changed till the end (96 h) of the culture. TCDD in the tissue was analysed by mass spectrometry, and the percentage of proliferating cells was measured using the MIB-1 labelling index. TCDD added to the culture medium accumulated in the tissues after 24 h (59.3%) and 96 h (81.2%) of exposure. The accumulative effect of TCDD was manifested by a reduction in the percentage of proliferating cells (53.5 and 33.8%, after 24 and 96 h exposure, respectively). A single exposure to TCDD had no effect on progesterone, reduced testosterone secretion and caused a significant increase in oestradiol secretion. Prolonged exposure to TCDD caused an increase in the concentration of the three steroids investigated in the culture medium. The results suggest that TCDD action is complex in the follicles.

Prognostic significance of DNA ploidy and proliferation rate in human astrocytic gliomas.

DNA ploidy and the proliferative potential in 75 gliomas were investigated using bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic nucleolar organizer regions (AgNOR) technique. There were 53 highly malignant (AIII-AIV), and 22 low-grade (AI-AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLI, tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with BrdUrd using the high pressure oxygen method. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. The same percentage of DNA aneuploidy (55%) was found in high-grade as well as in low-grade gliomas. Univariate analysis showed that patients with grade I & II gliomas had significantly higher 3-year survival rate (p = 0.0193) than those with grade III and grade IV gliomas. Also patients with lower proliferation rate of tumours (BrdUrdLI < or =2.3% and AgNOR counts < or =2.6%/cell) had higher 3-year survival rate (p<0.03), which can be helpful in prognosis. Tumour ploidy or SPF had no influence on patients' survival (p = 0.7908). Cox multivariate analysis showed that only patients' age > 45 years and high tumour grade (III and IV) were significant unfavourable prognostic factors in terms of patients' survival.

Differential anti-proliferative properties of novel hydroxydicarboxylatoplatinum(II) complexes with high or low reactivity with thiols.

We have examined the anti-proliferative effect of 13 recently synthesised platinum dicarboxylate complexes, very similar in their chemical, structural and kinetic properties to carboplatin. We used the L5178Y model: two murine lymphoma sublines, which differ in nucleotide excision repair ability and hence, in sensitivity to those platinum complexes that react with DNA. The anti-proliferative effect of the examined compounds mainly depends on the kind of amine ligand. Complexes with the primary amine (ethylenediamine) are more effective than complexes containing the tertiary amine (1-alkylimidazole). The ethylenediaminemalatoplatinum(II) complexes show a differential in vitro anti-proliferative activity in the L5178Y model; hence, it may be expected that they inflict DNA lesions that are repaired by the nucleotide excision system. The cytotoxicity of these complexes is directly correlated with reactivity with glutathione (GSH). The 1-alkylimidazole complexes are of low toxicity and moderate to low reactivity with GSH; in contrast to the ethylenediaminemalatoplatinum(II) complexes, their cytotoxicity is inversely correlated with reactivity with GSH. Two of the 1-alkylimidazole complexes, bis(1-ethylimidazole)(L-malato)platinum(II) and bis(1-propylimidazole (L-malato)platinum(II), show a considerable ability to arrest cells in G2 phase. We expect that the properties of these two groups of platinum complexes may be exploited in combined platinum complex treatment and irradiation.

Flow cytometric cell cycle analysis of two subpopulations of porcine granulosa cells.

Two types of granulosa cells from 120 individual follicles (forty follicles in particular stages of development) were analysed by DNA flow cytometry to determine the percentage of cells with degraded DNA (apoptosis) and the cell cycle analysis. The distribution of cells in the cell cycle (G1, S, G2/M) was studied to show relation to location within a follicle and follicular development. Isolated granulosa cells were scraped from the vesicle walls with rounded-tip ophthalmological tweezers and separated into weakly-associated (AGc) and tightly-bound (MGc) according to Ford (1991) and Duda (1997). Granulosa cells after fixation in 70% ethanol and staining with propidium iodide (PI) were analysed. At least 20,000 events were collected in each specimen. The S-phase fraction (SPF) and apoptosis were calculated using the ModFit LT programme for the cell cycle analysis (Verity Software House Inc., USA). In AGc a population with degraded DNA was found, containing less fluorescence than the G1/G0 peak as shown in the DNA histograms. The percentage of apoptotic AGc ranged from 39.29 in small, to 58.9 in medium and was significantly higher than in large follicles (26.13%; p < 0.05). The percentage of apoptotic MGc was significantly lower than in the AGc (p < 0.05) and was equal to 3.78 in small, 0.10 in medium and 0.08% in large follicles. There are no significant statistical differences between the mean percentage of SPF in MGc of small and medium follicles (4.94, 7.25%). However, SPF was significantly lower in large follicles (1.31%). The number of SPF in AGc decreased during follicular development (35.92, 26.98, and 19.62%). Our data indicate lack of apoptotic cell death in MGc which seem to be more differentiated, and lose their mitotic potential. In AGc however, which are undifferentiated and undergo numerous mitosis, apoptosis was observed.

Tumour cell kinetics as a prognostic factor in squamous cell carcinoma of the cervix treated with radiotherapy.

PURPOSE: Proliferative rate and DNA ploidy status were evaluated by flow cytometry in cervical cancer patients, prior to radiotherapy, to assess their importance as prognostic factors to predict survival rates. MATERIAL AND METHODS: Between 1987 and 1995, a total of 260 patients with squamous cell carcinoma (SCC) of the cervix, FIGO stages IB-IIIB were analysed. Tumour samples were incubated with bromodeoxyuridine (BrdUrd) in vitro to measure their total labelling index (totLI) and LI (totLI for diploid and anLI for aneuploid tumours). Proliferation was also assessed by S-phase fraction (SPF) analysis of the DNA profile. Patients had intracavitary therapy (three applications, each of 16 Gy to point A) and XRT of 40-50 Gy given over 4-5 weeks. RESULTS: The cervical tumours were characterized by a high proliferation rate which varied within each clinical stage of disease. The totLI ranged from 1.1 to 33.1% with median value of 9.6% whilst the LI ranged from 1.1 to 37.1% with a median value of 10.9%. Univariate analysis identified patient's age (cut-offpoint < or = 50&greater; years) and to a lesser extent proliferation (cut-off point, median totLI=9.6%) as significant prognostic factors for 5-year survival. The median survival time for younger patients ( < or = 50 years) with tumours of low proliferation (totLI < or = 9.6%) tumours was 17.5 months compared with 56 months in the faster proliferating tumours (P=0.0354). In the older patient sub-group, proliferation rate had no influence on survival. The median LI value was not a useful parameter in survival. Cox multivariate analysis showed that patient age ( < or = 50 years) and low proliferation of the tumour cells (totLI < or = 9.6) were unfavourable prognostic factors for cervical cancers treated with radiotherapy. DNA ploidy was not significant in this series. CONCLUSIONS: These data suggest that further improvements in therapy might be gained by selection of alternative treatments strategies such as neoadjuvant chemotherapy or radiation sensitizers in younger patients with more slowly proliferating tumours.

Flow cytometric analysis of DNA ploidy and proliferative potential in brain tumours.

The proliferative potential and DNA ploidy in 203 brain tumours (27 astrocytomas grade I, 37 grade II, 80 grade III, and 59 grade IV) were investigated using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry. One to three tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with bromodeoxyuridine (BrdUrd) using the high preasure oxygen method. After incubation, fixation and staining, the cell preparations were analysed by flow cytometry. The percentage of BrdUrd-labelled cells (BrdUrd Labelling Index, BrdUrdLI), the predicted potential doubling time (predTpot) and the total DNA content were evaluated. The percentage of unlabelled S-phase cells (SPF) and proliferative index (PI, the percentage of cells in S + G2 phases) were also estimated. DNA aneuploidy was found in 61.1% of high-grade (III-IV) and 50.0% of low-grade (I-II) astrocytomas. The tumours showed variability in the BrdUrdLI values which ranged from 0.2 to 15.8%. Significantly higher mean value for BrdUrdLI was shown in grade III-IV astrocytomas (3.4%), than in grade I-II astrocytomas (1.5%), p = 0.0068. Also significantly shorter mean predTpot was shown in high grade III-IV astrocytomas (28 days) than in low grade I-II tumours (51 days), p = 0.0096. However, no relationship was shown between other cell proliferation parameters and histological grade. The mean intratumoral variability calculated on the basis of BrdUrdLI values on 2-3 samples from each tumour amounted to 31.2% +/- SD 15.9%.

Tumour cell kinetics as prognostic factor in surgically-treated-non-small cell lung cancer.

The proliferative rate of tumour cells were studied in 80 non-small cell lung cancers (NSCLC) treated surgically at the Centre of Oncology in Kraków, between 1990 and 1996. There were 56 squamous cell carcinoma (SqLC) and 24 non-SqLC (18 adenocarcinoma (AcLC), three large cell carcinomas (LcLC), three mixed tumours). The proliferative potential of the tumour cells was studied on the basis of percentage of cells in the S-phase (S-phase fraction, SPF), proliferative index (PI, number of cells in S+ G2/M phases), bromodeoxyuridine labelling index (BrdUrdLI), and predictive potential doubling time (pred Tpot). Significant differences in the proliferating rate between histological groups of tumours were shown by the BrdUrdLI. The 5-year survival time for patients with higher proliferating tumours (BrdUrdLI > 4.1%, optimal cutoff level) was significantly higher (median survival time of > 60 months) than those with lower proliferative potential (BrdUrdLI < or = 4.1%) (median survival time of 19 months, P = 0.0091). SqLC patients had significantly better 5-year survival (median survival time of 47.5 months) than those with non-SqLC (median survival time of 18.5 months). Cox multivariate analysis showed that only higher proliferation of the tumour cells (BrdUrdLI > 4.1%), and lower clinical stage (I and II) were favourable prognostic factors in respect to patients' survival.

Clinical and flow cytometric prognostic factors in surgically treated squamous cell lung cancer.

A five-year follow-up study of prognostic factors in 207 patients with squamous cell lung cancer (SqLC) radically treated with surgery was investigated. Cellular prognostic indicators for survival times, such as percentage of cells in the S-phase (S-phase fraction, SPF), proliferative index (PI, number of cells in S + G2/M phases) and DNA ploidy, in addition to well known clinical factors were studied. Patients with aneuploid tumours had significantly shorter survival period (P < 0.05) than patients with diploid tumours. However, proliferative rate of the tumours had no influence on patients' survival. Cox multivariate analysis showed that metastases to the neighbouring lymph nodes, tumour diameter > 5 cm and DNA aneuploidy of the tumour cells were the negative factors which affected patients survival. DNA ploidy did not depend on the clinical stage of the tumours.

Lewis lung carcinoma and two transplantable sarcomas in mice as experimental therapy models: biological characteristics and cell population kinetics.

The biological characteristics of three transplantable tumours: two sarcomas (SaL, MCA) and Lewis lung carcinoma (LLC) have been studied. We investigated histology, DNA ploidy and cell kinetics parameters of the tumours. All examined tumours were aneuploid and rapidly proliferating, with the hyperdiploid fraction greater than 60%. The SaL tumour was found to have the lowest mean aneuploid bromodeoxyuridine labelling index (LI) equal to 21%, while the highest LI of 35.8% was measured for the LLC tumour. The mean S-phase time was short, lasting 8.5 - 10.9 hrs. The potential doubling time (T(pot)) assessed by BrdUrd staining and flow cytometry were as follows: 37.1 hours for SaL, 22.7 hours for MCA and 21.4 hours for LLC tumour. The MCA had the shortest volume doubling time (T(d)) equal to 1.7 days and the longest one, equal to 4.7 days, was found for the LLC. The lowest cell loss was found in the MCA tumour (44%), while the highest in the LLC tumour (81%). As all the examined tumours proliferate rapidly, there is a capacity for accelerated repopulation and therefore the tumours seem to be good models for experimental radiotherapy.

Intrinsic radiosensitivity of healthy donors and cancer patients as determined by the lymphocyte micronucleus assay.

The purpose of the study was to evaluate the usefulness of the cytokinesis-block micronucleus (MN) assay in assessment of radiosensitivity of lymphocytes in cancer patients. Lymphocytes from 15 cervical cancer patients, 21 head and neck cancer patients, seven lung cancer patients and 19 healthy donors were analysed using MN assay. The proportion of binucleate cells (BC) in cancer patients ranged from 22 to 56% and was significantly lower than in the control group (38-68%). MN frequency assessed five times over 6 months in four healthy donors showed that the interindividual variation was significantly higher than intraindividual. Before (0 Gy) and after irradiation (2 and 4 Gy) no statistical differences in the mean number of MN/BC were observed between healthy donors and cancer patient groups. Nevertheless, statistical cluster analysis allowed each group of donors to be divided into radioresistant and radiosensitive subgroups of patients. They showed significantly different dose response. Separate comparison of the mean MN frequency within all examined radioresistant and radiosensitive subgroups, showed statistically significant differences only after a dose of 4 Gy. At this dose, the lung cancer patients and cervical cancer patients from radiosensitive subgroups presented significantly higher radiosensitivity than the healthy donors. However, healthy donors from radioresistant subgroup did not differ significantly from cancer patients. This work has shown a high variation in interindividual radiosensitivity of donors and suggests the possibility of identifying radiosensitive patients on the basis of MN assay performed on lymphocytes.

Argyrophilic nucleolar organizer regions, bromodeoxyuridine labelling index and DNA ploidy in squamous cell carcinoma of the uterine cervix.

There is increasing evidence that rapidly proliferating tumours, i.e. those with a high bromodeoxyuridine labelling index (BrdUrdLI), could benefit from an accelerated course of radiotherapy. Also, DNA ploidy may be a prognostic factor in term of patients survival. Thus, measurements of cell kinetics and DNA ploidy might become part of routine characterization of tumours before treatment. It is supposed, that a simple and cheap argyrophilic nucleolar organizer regions (AgNOR) test reflects the proliferative status of the tumour and correlates with BrdUrdLI. The BrdUrdLI, AgNOR test and DNA ploidy were assessed in 49 squamous cell carcinoma (SCC) of the cervix (stage II B-III B) and 5 normal epithelium. The number of NORs per cell nucleus, the mean AgNOR particle area and the total AgNOR area per cell were evaluated. Significant differences in the proliferative rate were found within the examined groups of tumours assessed by the BrdUrdLI and AgNOR test. The mean BrdUrdLI values were significantly lower in normal than in carcinomatous cells, while for AgNOR values this was true for stage III B only. The mean number of AgNORs and total AgNOR area per cell were not significantly higher at stage III B than at stage II B, respectively. A high DNA aneuploidy was found in the examined tumours: 78% in stage II B and 77% in stage III B of disease. The results of proliferative markers were not significantly different in diploid than in aneuploid tumours. A significant correlation (p < 0.0001) was found between the mean AgNOR values and BrdUrdLI, however the correlation coefficient was poor (r = 0.50). This was due to different fragments of the same tumours used in these tests. Therefore these techniques might be used as independent methods reflecting the proliferative rate of the tumour.

Measurement of DNA ploidy and proliferation rate in lung tumours by bromodeoxyuridine labelling index, S-phase fraction and AgNOR technique.

Proliferative rate and DNA ploidy in 23 squamous cell carcinoma of the lung (SCCL), 10 adenocarcinomas (ACL) and 4 metastatic tumours (MT) were studied. The proliferative potential of the tumours was assessed with the use of three methods: bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic organizer regions (AgNOR) technique. Differences in the proliferating rate between different histological groups of tumours and within groups were shown by the BrdUrdLI and AgNOR technique. However, for the BrdUrdLI only these differences were statistically significant. No correlation was found between the values for all examined tests and the clinical stage of the tumours. The broadest range of the BrdUrdLI values was for SCCL and the mean BrdUrdLI was highest in this histological group. The number of SPF cells in metastatic tumours was lower than that in two other histological groups, however it was not a significant difference. The highest number of AgNORs and tot AgNOR area per cell was noticed again in SCCL, however, these values were not significantly different (p = 0.189) from other histological groups. Also the mean size of AgNOR particle area did not differ between groups (p = 0.279). Twenty six of thirty seven (70%) tumours were aneuploid. The highest percentage was recorded for SCCL (81%), while the lowest (25%) for MT. In the aneuploid tumours no significantly higher proliferation rate was observed as assessed by the BrdUrdLI and SPF. However, higher AgNOR numbers (p = 0.0472) and higher tot AgNOR area per cell (p = 0.0128) in aneuploid than in diploid tumours were observed. There was no correlation between BrdUrdLI, SPF and AgNOR counts.

Effects of topoisomerase I-targeted drugs on radiation response of L5178Y sublines differentially radiation and drug sensitive.

The murine L5178Y (LY) lymphoma sublines, LY-R (radiation resistant) and LY-S (radiation sensitive) display a difference in susceptibility to camptothecin (CPT): LY-S cells are less sensitive to killing by this inhibitor of topoisomerase I than LY-R cells. Post-treatment (CPT present until 3 h after irradiation) sensitizes only LY-S cells. In agreement with this, only in LY-S cells is the relative number of DNA-protein cross-links formed after treatment with CPT + X higher than expected for additivity of X-ray and CPT-induced damage. The pattern of changes in the labelling indices and cell cycle distribution in cells that underwent combined treatment is essentially like that seen for single-agent treatment: for LY-S cells like that for radiation, for LY-R cells like that for CPT. We found no direct relation between the patterns of cell cycle distributions and the enhancement of the lethal effect of X-irradiation by CPT post-treatment. The sublines are not markedly differentially sensitive to beta-lapachone, which modifies topoisomerase I activity, and not sensitized to X-rays by post-irradiation treatment with the drug.

Micronucleus assay in three transplantable mouse tumors.

The cytokinesis-block micronucleus (MN) assay was performed in three mouse tumors: two sarcomas (SaL, MCA) and Lewis lung carcinoma (LLC). To determine the optimal culture durations and cytochalasin B (cyt-B) concentrations to yield the highest proportion of binucleate cells (BC) for each tumor, the influence of the cyt-B concentration (1, 2 and 3 micrograms/ml) and culture duration (24-96) were studied. The amount of BC and the MN frequency was investigated for the different radiation doses (0-4 Gy). Dose response curves were constructed using the optimal culture duration and cyt-B concentration for each tumor. This was 24 h of incubation for MCA and 48 h for SaL and LLC and 2 micrograms/ml of cyt-B. The tumors examined differ in the mean number of spontaneously (0 Gy) occurring MN in binucleate cells. These were 0.008, 0.022 and 0.044 for MCA, SaL and LLC, respectively. The MN frequency increased with radiation dose. LLC was found to be the most radiosensitive, while MCA proved to be the least radiosensitive tumor. The average number of MN/BC at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.05 to 0.36. The highest mean value -0.36 was shown in LLC, the middle-0.20 in SaL, and the lowest-0.05 in MCA tumor. After higher doses of irradiation numerous BC with two and more MN were found in LLC tumor, while they were not frequently observed in MCA tumor. We did not observe an increase in the MN frequency with culture duration or proliferation rate of the tumor cells. MCA has the shortest potential doubling time (Tpot) and had the lowest MN frequency from three examined tumors. The MN assay has promise to be a rapid predictive assay of radiosensitivity.